CN113310958B - Preparation method of hierarchical porous metal organic framework chiral sensing probe, probe obtained by preparation method and application of probe - Google Patents
Preparation method of hierarchical porous metal organic framework chiral sensing probe, probe obtained by preparation method and application of probe Download PDFInfo
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Abstract
The invention relates to a preparation method of a hierarchical porous metal organic framework chiral sensing probe, which comprises the steps of providing a hierarchical porous metal organic framework microsphere with a regular pore channel structure; fixing amino acid oxidase on the hierarchical porous metal organic framework microsphere to obtain the hierarchical porous metal organic framework microsphere with the enzyme fixed, wherein the amino acid oxidase is loaded in a pore channel of the hierarchical porous metal organic framework microsphere through adsorption; and (3) fixing the fluorescent molecules on the hierarchical porous metal organic framework microspheres fixed with the enzyme to obtain the hierarchical porous metal organic framework chiral sensing probe, wherein the fluorescent molecules are loaded in the pore channels of the hierarchical porous metal organic framework microspheres through adsorption. The invention also provides a hierarchical porous metal organic framework chiral sensing probe obtained by the preparation method and application thereof. The invention can identify and analyze the content of amino acid with high sensitivity and high specificity by fixing different amino acid oxidases as chiral identification centers.
Description
Technical Field
The invention relates to enzyme immobilization and analytical chemistry, in particular to a preparation method of a hierarchical porous metal organic framework chiral sensing probe, the probe obtained by the method and application of the probe.
Background
Amino acids are among the most predominant and important chiral compounds in nature (Angew. Chem. Int. Ed.2017,56, 7276-7281). L-amino acids and D-amino acids are not mutually exclusive and generally coexist in non-racemic mixtures. In mammals and humans, amino acids are usually present in free form or in the form of proteins. In general, most amino acids in organisms and nature exist in L form, while the abundant presence of D form usually indicates negative symptoms, senescence or disease (ACS appl. The production of Amino Acids, racemization, is of great value in life sciences (Amino Acids 2012,42, 1553-1582). Amino acid enantiomers are also widely used as chiral sources of asymmetry. The total amount of amino acids in the central nervous system, as well as the enantiomeric ratio, often exhibit different biological functions, playing a crucial role in human physiology and pathology, and the expression levels of specific chiral amino acids in biological systems are often associated with early stages of many diseases, such as chronic kidney disease, alzheimer's disease and cancer (j.am. Chem. Soc.2016,138, 12099-12111). Therefore, the method has important significance for rapidly and accurately carrying out chiral analysis on the amino acid. To date, there are various methods for selective recognition and detection of amino acid enantiomers, including chromatography, capillary electrophoresis, fluorescence, circular dichroism, and the like (biosens. Bioelectronic. 2020,151, 111971). However, conventional chromatography, capillary electrophoresis, and the like for enantiomer recognition and separation are effective for chiral detection of amino acids, but the test cost is high, the procedures and steps required for the test are cumbersome, and the detection time is long. The fluorescent sensor can avoid the defects and can quickly, effectively, simply and conveniently detect the chiral amino acid. The method is suitable for high-throughput screening and real-time imaging of amino acid enantiomers in biological samples. However, the design of chiral binding/reaction sites is a key and enormous challenge for enantioselective recognition of fluorescent probes.
To achieve a stereochemical interaction between a substrate and a probe molecule requires a complex and precise chemical synthesis process. Meanwhile, fluorescent probes rarely simultaneously enantioselectively and chemoselectively recognize specific amino acids and may be interfered by other similar biochemical molecules. Therefore, the development of a general fluorescence sensing strategy suitable for broad-spectrum detection of chiral amino acids, whether enantioselectivity or chemoselectivity, is of great significance.
Disclosure of Invention
In order to realize enantioselective and chemoselective recognition of amino acid by a fluorescent probe, the invention provides a preparation method of a hierarchical porous metal organic framework chiral sensing probe, the probe obtained by the preparation method and application of the probe.
The preparation method of the hierarchical porous metal organic framework chiral sensing probe comprises the following steps: s1, providing a hierarchical porous metal organic framework microsphere (HPUiO-66) which has a regular pore channel structure; s2, fixing Amino Acid Oxidase (AAO) on the hierarchical porous metal organic framework microsphere (HPUiO-66) to obtain the hierarchical porous metal organic framework microsphere (AAO @ HPUiO-66) fixed with the enzyme, wherein the Amino Acid Oxidase (AAO) is loaded in a pore channel of the hierarchical porous metal organic framework microsphere (HPUiO-66) through adsorption; s3, fixing the fluorescent molecule (PF) on the hierarchical porous metal organic framework microsphere (AAO @ HPUiO-66) fixed with the enzyme to obtain the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66), wherein the fluorescent molecule (PF) is loaded in the pore channel of the hierarchical porous metal organic framework microsphere (HPUiO-66) through adsorption.
Preferably, the Amino Acid Oxidase (AAO) is an amino acid oxidase having a chiral recognition function. More preferably, the Amino Acid Oxidase (AAO) is at least one selected from the group consisting of an L-amino acid oxidase, an L-glutamate oxidase, and an L-tryptophan oxidase.
Preferably, the fluorescent molecule (PF) is of H 2 O 2 A responsive fluorescent molecule. More preferably, the fluorescent molecule (PF) is 3-Oxo-3',6' -bis (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -3H-spiro [ isobenzofuran-1, 9' -ton]-6-carboxylic acid.
Preferably, the step S1 includes: s11, by means of the Block copolymer PEO 106 PPO 70 PEO 106 (F127) Providing a Metal Organic Framework (MOFs) precursor solution for a template agent; s12, generating a hierarchical porous metal organic framework precursor (HPUiO-66-as) through solvothermal reaction; s13, obtaining the hierarchical porous metal organic framework microsphere (HPUiO-66) with large pore diameter by activating and removing the template. More preferably, the pore size of the hierarchical porous metal organic framework microsphere (HPUiO-66) is 8-10nm. In a preferred embodiment, the pore size of the hierarchical porous metal organic framework microsphere (HPUiO-66) is 9.2nm. More preferably, the block copolymer F127 is dissolved in deionized water under stirring, sodium perchlorate and acetic acid are added, and ultrasonic dissolution is carried out; then cerium ammonium nitrate and terephthalic acid are added, and stirring and dispersing are carried out; stirring and reacting for 20min at 60 ℃; and obtaining the graded porous metal organic framework microsphere (HPUiO-66) after centrifugal separation, washing, activation and drying. Specifically, 150mg of F127 was dissolved in 9mL of deionized water, followed by 750mg of NaClO 4 ·H 2 O and 450 mu L of acetic acid are ultrasonically dispersed in the F127 solution; subsequently, 249mg of terephthalic acid and 274mg of cerium ammonium nitrate were dispersed in the above solution with stirring; then, the mixed liquid is stirred and reacted for 20 minutes at the temperature of 60 ℃; centrifuging the mixture to obtain a solid product; then deionized water, N-dimethylformamide and anhydrous ethyl acetate are usedWashing the material with alcohol, soaking the material in absolute ethyl alcohol at 60 ℃ for 48h, continuously replacing the alcohol during the 48h, completely removing F127 in the pore canal of the material, and finally obtaining the hierarchical porous metal organic framework microsphere (HPUiO-66) with the size of 600 nm-1000 nm after washing and drying. The invention uses the block copolymer F127 as a template and uses
The Hofmeister salt-soluble mediated effect is adopted to synthesize the hierarchical porous metal organic framework microsphere (HPUiO-66) with large aperture of about 9nm, the synthesis steps are simple, and the preparation period is short.
Preferably, the step S2 includes: dissolving Amino Acid Oxidase (AAO) with chiral catalytic function in deionized water, adding hierarchical porous metal organic framework microspheres (HPUiO-66), stirring at 25 ℃ for 4h, and collecting the hierarchical porous metal organic framework microspheres (AAO @ HPUiO-66) immobilized with enzyme by centrifugation.
Preferably, the step S3 includes: dissolving fluorescent molecules (PF) in deionized water, adding hierarchical porous metal organic framework microspheres (AAO @ HPUiO-66) immobilized with enzyme, stirring at 25 deg.C in dark place for 30min, and centrifuging to collect hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66).
In the invention, amino Acid Oxidase (AAO) is a natural enzyme, fluorescent molecules (PF) are fluorescent small molecules, and the amino acid oxidase and the fluorescent small molecules are sequentially fixed in mesoporous channels of hierarchical porous metal organic framework microspheres (HPUiO-66) by a mild post-loading method to prepare a series of fluorescent probes with high specific sensing performance. Specifically, at room temperature, amino Acid Oxidase (AAO) and fluorescent molecule (PF) are sequentially fixed in hierarchical mesopores of hierarchical porous metal organic framework microspheres (HPUiO-66) in a post-adsorption mode to synthesize a hierarchical porous metal organic framework chiral sensing probe (AAO)&PF @ HPUiO-66), amino Acid Oxidase (AAO) loading was 151mg g -1 The immobilized amount of fluorescent molecule (PF) was 374mg g -1 The preparation process has mild conditions and simple and convenient operation.
The invention also provides a hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) obtained by the preparation method, wherein Amino Acid Oxidase (AAO) and fluorescent molecule (PF) are coupled on a carrier of a hierarchical porous metal organic framework microsphere (HPUiO-66).
The invention also provides application of the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66), which has chiral sensing response to amino acid. Therefore, the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) is used for identifying chiral amino acid through a cascade response system constructed by Amino Acid Oxidase (AAO) and fluorescent molecules (PF).
Preferably, the amino acid is phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine, and/or cysteine.
Preferably, the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) shows fluorescence enhancement for the L-enantiomer of the amino acid, but no response to the D-enantiomer of the amino acid.
Preferably, the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) is dispersed in HEPES buffer solution to form a probe solution, an amino acid solution is added into the probe solution for incubation, and the fluorescence intensity is measured by a fluorescence spectrometer.
Preferably, the amino acid is linearly analyzed by using a hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) in a concentration range of 0 to 100. Mu.M in the amino acid solution. Accordingly, the hierarchical porous metal organic framework chiral sensing probe (AAO & PF @ HPUiO-66) can be used for quantitative analysis of chiral amino acid.
Preferably, the lower limit of detection of the amino acid solution is 0.38. Mu.M to 0.44. Mu.M.
According to the invention, the enzyme and the fluorescent molecules are sequentially loaded into the hierarchical porous metal organic framework in a post-fixing mode, the firm exoskeleton of the metal organic framework material protects the protein structure of the enzyme, the stability of the enzyme is improved, and the hierarchical porous structure also fixes the fluorescent molecules on the metal nodes in a separated manner, so that aggregation and quenching of the fluorescent molecules are avoided; the large pore diameter and the high porosity of the material are beneficial to the rapid diffusion of the material andreaction, in the course of detection cascade reaction is used, i.e. oxidation deamination reaction between L-amino acid oxidase and L-amino acid is used to produce H 2 O 2 Followed by H 2 O 2 The chiral amino acid is subjected to ring-opening reaction with a fluorescent molecule PF to generate strong fluorescence, and a fluorescence spectrophotometer is used for detection, so that the aim of chiral amino acid detection is fulfilled. The preparation method has high ductility, realizes chiral recognition and quantitative analysis of different amino acids by loading different natural amino acid oxidases, provides a simple and effective way for designing enantioselective and chemoselective fluorescence sensors, and has important significance for realizing rapid and accurate chiral recognition and quantitative analysis of amino acids. According to the invention, different amino acid oxidases are fixed to serve as chiral recognition centers, so that chiral amino acids can be recognized and subjected to content analysis with high sensitivity and high specificity. The method has the advantages of convenient synthesis method, mild preparation conditions, convenient instrument operation, low technical requirement, high analysis sensitivity, strong specificity and excellent anti-interference capability. The hierarchical porous metal organic framework chiral sensing probe can specifically and selectively identify the L-enantiomer of 8 amino acids, namely phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine and cysteine, can perform linear determination on the L-amino acid in a low concentration range of less than 100 mu M, and has the advantages of low detection limit (0.38 mu M-0.44 mu M), high sensitivity, strong specificity and excellent anti-interference capability; and the detection instrument is simple, the operation is convenient, and the technical requirement is low. The strategy provided by the invention has universal applicability, and can simply replace L-amino acid oxidase with L-glutamate oxidase, so that the selective identification and content analysis of free L-glutamate in an aqueous phase can be realized. And also has high specificity, low detection limit and strong anti-interference capability.
Drawings
FIG. 1 is a process flow diagram of a method for preparing a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of fluorescent molecules (PF) of the hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 3 is a graph of adsorption kinetics of a hierarchical porous metal organic framework chiral sensing probe when loaded with L-amino acid oxidase L-AAO and a fluorescent molecule PF according to the present invention;
FIG. 4 is an XRD pattern of a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 5 is a scanning electron microscope image of a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 6 is a nitrogen adsorption diagram and its pore size distribution diagram of a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 7 is two enantiomeric kinetic response profiles of a hierarchical porous metal organic framework chiral sensing probe to phenylalanine according to the present invention;
FIG. 8 is two enantiomeric kinetic response profiles of a hierarchical porous metal organic framework chiral sensing probe to leucine according to the present invention;
FIG. 9 is two enantiomeric kinetic response profiles of a hierarchical porous metal organic framework chiral sensing probe to methionine according to the present invention;
FIG. 10 is two enantiomeric kinetic response profiles of a hierarchical porous metal organic framework chiral sensing probe according to the present invention to tryptophan;
FIG. 11 is two enantiomeric kinetic response profiles of a hierarchical porous metal organic framework chiral sensing probe to histidine according to the present invention;
FIG. 12 is a graph of two enantiomeric kinetic responses of a hierarchical porous metal organic framework chiral sensing probe for isoleucine according to the present invention;
FIG. 13 is a graph of two enantiomeric dynamic response of a hierarchical porous metal organic framework chiral sensing probe to tyrosine, according to the present invention;
FIG. 14 is two enantiomeric kinetic response spectra of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for cysteine;
FIG. 15 is a fluorescence titration spectrum of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for the L-enantiomer of phenylalanine;
FIG. 16 is a fluorescence titration spectrum of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for the L-enantiomer of leucine;
FIG. 17 is a fluorescence titration spectrum of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for the L-enantiomer of methionine;
FIG. 18 is a fluorescence titration spectrum of L-enantiomer of tryptophan with a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 19 is a fluorescence titration spectrum of L-enantiomer of histidine with a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 20 is a fluorescence titration spectrum of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for the L-enantiomer of isoleucine;
FIG. 21 is a fluorescence titration spectrum of a hierarchical porous metal organic framework chiral sensing probe according to the present invention for the L-enantiomer of tyrosine;
FIG. 22 is a fluorescence titration spectrum of L-enantiomer of cysteine with a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 23 is an anti-interference, selective detection result image of a hierarchical porous metal organic framework chiral sensing probe according to the present invention;
FIG. 24 is a graph showing fluorescence kinetics curves of two enantiomeric responses of a hierarchical porous metal organic framework chiral sensing probe to glutamic acid, a fluorescence titration spectrum for L-glutamic acid, and a selective detection result image for L-glutamic acid according to the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the preparation method of the hierarchical porous metal organic framework chiral sensing probe according to the present invention comprises the steps of: providing MOFs precursor solution; generating a hierarchical porous UiO-66 precursor (HPUiO-66-as) by solvothermal; obtaining graded porous UiO-66 nano particles (HPUiO-66) by activating and removing a template; fixing L-amino acid oxidase (L-AAO) on the hierarchical porous UiO-66 nano particles (HPUiO-66) to obtain an enzyme-fixed hierarchical porous metal organic framework (L-AAO @ HPUiO-66), wherein the L-amino acid oxidase (L-AAO) is loaded in the pore channels of the hierarchical porous UiO-66 nano particles (HPUiO-66) through adsorption; and (2) fixing a fluorescent molecule (PF) on a hierarchical porous metal organic framework (L-AAO @ HPUiO-66) immobilized with enzyme to obtain a hierarchical porous metal organic framework chiral sensing probe (L-AAO & PF @ HPUiO-66), wherein the fluorescent molecule (PF) is loaded in a pore channel of the hierarchical porous UiO-66 nanoparticle (HPUiO-66) through adsorption.
Example 1
1.1 Synthesis of fluorescent molecules (PF)
1,2, 4-benzenetricarboxylic acid having a mass of 3.15g and 3-bromophenol having a mass of 5.19g and methanesulfonic acid having a volume amount of 15mL were added to a three-necked flask. And heated at 135 deg.c under reflux for 72 hours, after cooling naturally, the mixture was added to 120mL of ice deionized water, and stirred to give a green solid, and then the resulting solid was collected by vacuum filtration. And washed several times with a mixed reagent of pyridine and acetic anhydride (v/v = 1), and recrystallized 3 times with a mixed reagent of acetic anhydride and pyridine (v/v =2 -1 The hydrochloric acid washing several times, finally, at 85 degrees C through vacuum drying for 12h get white solid powder.
400mg of bis (pinacol) diboron, 200mg of the above solid powder, 98.9mg of Pd (dppf) Cl 2 And anhydrous potassium acetate having a mass of 510mg were charged into a dry three-necked flask, and the system was evacuated and then charged with nitrogen again. This process is repeated at least three times. Then, anhydrous, degassed N, N-dimethylformamide was added in a volume amount of 5 mL. The mixed solution was then stirred at room temperature for 5 minutes and then heated at 85 ℃ under reflux for 3 hours. After the reaction was finished and naturally cooled, the solvent was removed by rotary evaporation, and the crude product was purified by gradient washing with a mixed solvent of dichloromethane and methanol through a silica gel column equilibrated with dichloromethane, then a minimum amount of anhydrous diethyl ether was added dropwise to precipitate a light brown solid, and washed several times with anhydrous diethyl ether, and finally dried in vacuo at 40 ℃Drying for 12h to obtain bone white Powder (PF).
The nmr hydrogen spectrum of fig. 2 demonstrates the successful synthesis of PF.
1.2 Synthesis of hierarchical porous UiO-66 nanoparticles
F127 with a mass of 150mg was dissolved in 9mL of deionized water at room temperature, followed by NaClO with a mass of 750mg 4 ·H 2 O and acetic acid in a volume of 450. Mu.L were ultrasonically dispersed in the F127 solution described above. Subsequently, terephthalic acid having a mass of 249mg and cerium ammonium nitrate having a mass of 274mg were dispersed in the above solution with stirring. Then, the mixed liquid was stirred at 60 ℃ to react for 20 minutes. The mixture was centrifuged to give a solid product. And then washing the material with deionized water, N-dimethylformamide and absolute ethyl alcohol, soaking the material in the absolute ethyl alcohol at 60 ℃ for 48 hours, continuously replacing the ethyl alcohol in the process, completely removing F127 in pore channels of the material, and finally obtaining the hierarchical porous metal organic framework material (HPUiO-66) with the size of 700 nm-800 nm after washing and drying.
1.3 immobilization of L-amino acid oxidase (L-AAO) to obtain an enzyme-immobilized hierarchical porous Metal organic framework (L-AAO @)
HPUiO-66)
L-amino acid oxidase, 2.5mg in mass, was dissolved in 2.5mL of deionized water, and then HPUiO-66 nanoparticles, 2.5mg in mass, were dispersed in the enzyme solution. And stirred at 25 ℃ for 4 hours. The enzyme-immobilized HPUiO-66 material (L-AAO @ HPUiO-66) was collected by centrifugation and washed several times with deionized water to remove the enzyme adsorbed on the surface of HPUiO-66.
1.4 fixing fluorescent molecules (PF) to obtain a hierarchical porous metal organic framework chiral sensing probe (L-AAO)&PF@
HPUiO-66)
Fluorescent molecule PF with a mass of 5mg was dissolved in 5mL of deionized water, and then L-AAO @ HPUiO-66 nanoparticles with a mass of 2.5mg were dispersed in the above solution. And stirred at 25 ℃ for 30min in the dark. The HPUiO-66 material (L-AAO & PF @ HPUiO-66) immobilized with enzyme and fluorescent molecule was collected by centrifugation and washed several times with deionized water to remove PF adsorbed on the surface.
FIG. 3 is a graph showing the adsorption kinetics of L-amino acid oxidase L-AAO and fluorescent molecule PF. FIG. 4 is an XRD pattern of HPUiO-66 and L-AAO & PF @ HPUiO-66, it can be seen that the characteristic peak and high crystallinity of UiO-66 crystal are maintained no matter the hierarchical porous metal organic framework material HPUiO-66 or the hierarchical porous metal organic framework chiral probe material L-AAO & PF @ HPUiO-66; FIG. 5 is a scanning electron microscope image of HPUiO-66 (A) and L-AAO & PF @ HPUiO-66 (B), which shows that HPUiO-66 and L-AAO & PF @ HPUiO-66 both have regular channel structure and particle size of 700 nm-800 nm. FIG. 6 is a nitrogen adsorption-desorption curve and pore size distribution of HPUiO-66 and L-AAO & PF @ HPUiO-66. It can be seen that L-AAO & PF @ HPUiO-66 has a significantly reduced specific surface area, porosity and pore size compared to HPUiO-66. It was confirmed that L-AAO and PF were loaded into the channels of HPUiO-66, rather than adsorbed on the surface. The pore structure parameters for HPUiO-66 and L-AAO & PF @ HPUiO-66 are given in Table 1 below.
TABLE 1
| Sample (I) | Aperture (nm) | Pore volume (cm) 3 /g) | Specific surface area (m) 2 /g) |
| HPUiO-66 | 9.2 | 0.626 | 1217.3 |
| L-AAO&PF@HPUiO-66 | 6.7 | 0.142 | 262.0 |
1.5 hierarchical porous metal organic framework chiral sensing probe (L-AAO)&PF @ HPUiO-66) chirality of amino acids
Identification
The mass is 5mg of L-AAO&PF @ HPUiO-66 Probe Material dispersed in 50mL of HEPES buffer (20mM, pH 7.4) to form 100mg of L -1 The probe solution of (4), 100. Mu.L of an L/D-amino acid solution (2 mM L) -1 ) The resulting mixture was added to 1.9mL of the above probe solution, and the reaction was incubated at 37 ℃ under exclusion of light. Then, the fluorescence intensity of the mixture at λ =520nm was detected at fixed time intervals. The L-AAO can be seen from the images of FIGS. 7-14&The PF @ HPUiO-66 probe has a chiral sensory response to 8 amino acids (phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine, cysteine). Shows an increase in fluorescence for the L-enantiomer, but no response to the corresponding D-enantiomer.
1.6 hierarchical porous metal organic framework chiral sensing probe (L-AAO)&PF @ HPUiO-66) content of amino acids
Analysis of
The mass is 5mg of L-AAO&PF @ HPUiO-66 Probe Material dispersed in 50mL of HEPES buffer (20mM, pH 7.4) to form 100mg of L -1 The probe solution of (1), 100 μ L of L-amino acid solutions of different concentrations were added to 1.9mL of the above probe solution, the reaction was incubated at 37 ℃ in the dark until equilibrium, and the fluorescence intensity of the above reaction mixture at λ =520nm was measured by a fluorescence spectrometer. As can be seen from the images of FIGS. 15 to 22, when the L-enantiomers of the above 8 amino acids were added, the fluorescence intensity of the probe solution was regularly increased with the increase in the concentration of the L-amino acid, and the probe could perform linear analysis of the L-amino acid in the concentration range of 0 to 100. Mu.M, with a correlation coefficient as high as 0.99 and a lower detection limit LOD (0.38. Mu.M to 0.44. Mu.M). The following Table 2 shows the fluorescence detection characteristics of the probe for the above 8L-amino acidsAnd (5) summarizing sex.
TABLE 2
1.7 hierarchical porous Metal organic framework chiral sensing Probe (L-AAO)&PF @ HPUiO-66) in interfering with ionization neutralization
Fluorescence sensing detection of amino acids in the presence of compounds
Will interfere with the substance (Na) + ，K + ，Mg 2+ ，Al 3+ ，Br - ，Cl - ，HCO 3 - ，NO 2 - Glucose (GLU), glutathione (GSH), cholesterol (CHOL)) was dissolved in the probe solution at a concentration of 100. Mu.M, and the probe solution was subjected to fluorometry before and after addition of 100. Mu.L L-phenylalanine (100. Mu.M). Fluorescence intensity at λ =520nm was obtained. In FIG. 23, A shows that the above-mentioned interfering substance has a negligible effect on the fluorescence characteristics of the probe itself, and B shows that the interfering substance has no effect on the detection result of the L-amino acid when it is present in the mixed solution to be detected.
1.8 hierarchical porous metal organic framework chiral sensing probe (L-AAO)&PF @ HPUiO-66) in a simulated physiologic ring
Fluorescence sensing detection of environmental amino acids
Under simulated physiological conditions, i.e., L-phenylalanine and D-phenylalanine, and L-AAO&PF @ HPUiO-66 probe material is dissolved in Simulated Body Fluid (SBF), wherein, the SBF solution preparation method is as follows: naCl and NaHCO 3 ，KCl，K 2 HPO 4 ，MgCl 2 ，CaCl 2 ，Na 2 SO 4 Dissolving in deionized water, and adding inorganic ions (mM: na) + 142，K + 5，Mg 2+ 1.5，Ca 2+ 2.5，Cl - 149，
18，
) Corresponding to human plasma concentration, using tris- (hydroxymethyl) aminomethane [ (CH) at 37 deg.C 2 OH) 3 CNH 2 ]And hydrochloric acid (HCl) to adjust the buffered pH of the liquid to 7.4. And according to1.6 points of Hierarchical porous metal organic framework chiral sensing probe (L-AAO)&PF @ HPUiO-66) content analysis of amino acidsThe test process of (1) is to perform selective sensing of L-phenylalanine in mixed solutions of L-phenylalanine and D-phenylalanine with different concentrations and perform four groups of parallel experiments simultaneously to obtain the average concentration. The results in Table 3 below demonstrate that L-AAO&The PF @ HPUiO-66 probe mimics the sensing ability of L-phenylalanine under physiological conditions.
TABLE 3
| Sample (I) | Additive concentration (μ M) | Determination of the concentration (. Mu.M). + -. Sigma | Recovery (%) |
| L-Phe/D- |
5/100 | 5.2±0.3 | 104±5 |
| L-Phe/D- |
20/100 | 19.6±0.6 | 98±3 |
| L-Phe/D- |
40/100 | 41.6±0.5 | 104±1 |
| L-Phe/D- |
60/100 | 60.4±1.0 | 101±2 |
Example 2
With reference to example 11.2-1.4Changing the fixed amino acid oxidase, and fixing the L-glutamate oxidase (L-GOX) into the hierarchical mesoporous metal organic framework microspheres to obtain the L-GOX&PF @ HPUiO-66 probe material.
See example 1 for1.5The L/D-amino acid is changed into L/D-glutamic acid. FIG. 24A shows L-GOX&The PF @ HPUiO-66 probe has chiral sensing ability for L-glutamic acid.
See example 1 for1.6Changing L-AAO&PF @ HPUiO-66 probe material is L-GOX&PF @ HPUiO-66 probe material; changing the L-amino acid into L-glutamic acid. L-GOX can be seen in FIGS. 24B and 24C&The PF @ HPUiO-66 probe can perform linear analysis on L-glutamic acid within 0-100. Mu.M.
100. Mu.L of a solution of 19 chiral enantiomers of amino acids (100. Mu.M) was added to 1.9mL of L-GOX&PF @ HPUiO-66 Probe solution (100 mg L) -1 ) In (1). The reaction mixture was incubated at 37 ℃ with exclusion of light until equilibrium and the fluorescence intensity of the reaction mixture at λ =520nm was measured using a fluorescence spectrometer. FIG. 24D shows that the probe has high selectivity for L-glutamic acid and no response to other L-amino acids and D-amino acids.
See example 1 for1.8And the L-phenylalanine and the D-phenylalanine are changed into L-phenylalanine, L-leucine, L-methionine, L-tryptophan, L-histidine, L-isoleucine, L-tyrosine, L-cysteine and D-glutamic acid. The results in Table 4 below demonstrate L-GOX&The PF @ HPUiO-66 probe mimics the sensing ability of L-glutamic acid under physiological conditions.
TABLE 4
The invention is based on the use of a block copolymer F127 template and
and synthesizing the hierarchical porous metal organic framework material by the Hofmeister salinization anion action. And loading different types of amino acid oxidase and fluorescent molecules into the pore channels of the hierarchical porous metal organic framework material in a post-adsorption mode, thereby realizing the chiral recognition and content analysis of the amino acid. The method has universal applicability, and provides a new method and thought for biomolecule fluorescence sensing.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications may be made to the above-described embodiment of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present invention are within the scope of the claims of the present invention. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (9)
1. A preparation method of a hierarchical porous metal organic framework chiral sensing probe is characterized by comprising the following steps:
s1, providing a hierarchical porous metal organic framework microsphere which has a regular pore channel structure;
s2, fixing amino acid oxidase on the hierarchical porous metal organic framework microsphere to obtain the hierarchical porous metal organic framework microsphere fixed with the enzyme, wherein the amino acid oxidase is selected from at least one of the group consisting of L-amino acid oxidase, L-glutamate oxidase and L-tryptophan oxidase, and is loaded in a pore channel of the hierarchical porous metal organic framework microsphere through adsorption;
s3, fixing a fluorescent molecule on the hierarchical porous metal organic framework microsphere with the immobilized enzyme to obtain the hierarchical porous metal organic framework chiral sensing probe, wherein the fluorescent molecule is 3-Oxo-3',6' -bis (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -3H-spiro [ isobenzofuran-1, 9' -ton ] -6-carboxylic acid, and is loaded in a pore channel of the hierarchical porous metal organic framework microsphere through adsorption.
2. The method according to claim 1, wherein the step S1 includes:
s11, using Block copolymer PEO 106 PPO 70 PEO 106 Providing a metal organic framework precursor solution for a template agent;
s12, generating a hierarchical porous metal organic framework precursor through solvothermal reaction;
and S13, obtaining the hierarchical porous metal organic framework microspheres with large pore diameters by activating and removing the template.
3. The chiral sensing probe with the hierarchical porous metal organic framework, which is obtained by the preparation method according to claim 1 or 2, is characterized in that amino acid oxidase and fluorescent molecules are coupled on a carrier of microspheres with the hierarchical porous metal organic framework.
4. The use of the hierarchical porous metal organic framework chiral sensing probe according to claim 3, wherein the hierarchical porous metal organic framework chiral sensing probe has a chiral sensing response to an amino acid.
5. The use according to claim 4, wherein the amino acid is phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine, and/or cysteine.
6. The use according to claim 4, wherein the hierarchical porous metal organic framework chiral sensing probe exhibits an increase in fluorescence for the L-enantiomer of the amino acid and no response to the D-enantiomer of the amino acid.
7. The use according to claim 4, wherein the hierarchical porous metal organic framework chiral sensing probe is dispersed in HEPES buffer solution to form a probe solution, an amino acid solution is added into the probe solution for incubation, and the fluorescence intensity is measured by a fluorescence spectrometer.
8. The use according to claim 7, characterized in that the amino acids are analyzed linearly with a hierarchical porous metal organic framework chiral sensor probe in the concentration range of 0 to 100 μ M in the amino acid solution.
9. The use according to claim 7, wherein the lower limit of detection of the amino acid solution is 0.38 μ M to 0.44 μ M.
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